MembranePro Functional Protein Expression
Contents
1. 21 7 By default Result and Images are saved as JPEG files To choose a different file format press the file type button Choose file type screen opens Note Data can only be saved as a CSV file 8 Press to select the desired file type Available options are JPEG BMP PNG and TIFF Choose file type After you make your selection the instrument returns to the Save screen To return to the Save screen without changing the file format press the close button 9 Press Save to save your experiment in the selected mode s in the USB drive Save complete Save complete 10 Press Close and then transfer the USB drive to the desired location 22 6 Instrument settings Overview Instrument Settings To access the Instrument Settings screen press the Instrument Settings screen button on the Home page page 8 Instrument Settings Software Update Date Time Change Light Cube In the Instrument Settings screen you can e perform software update page 24 e set the date and time page 25 dy Note Although the Change Light Cube button is visible on the Instrument Settings screen this button is not functional on the Countess II Automated Cell Counter which is a brightfield only instrument and does not support EVOS light cubes 29 Software update Guidelines for e The USB drive used for transferring the software update file must be FAT32 software update formatted verify2. Do not press the main plug or power cord with wet hands Always ensure that the power supply input voltage matches the voltage available in your location Do not install the instrument on a slant or a place prone to vibrations which induces the risk of instrument malfunction or damage of the instrument Never insert any objects into the air vents of the instrument as this could result in electrical shock personal injury and equipment damage Plug the power cord firmly into the wall outlet and the instrument To avoid potential shock hazard make sure that the power cord is properly grounded Be sure to position the equipment such that it is easy to disconnect the instrument Turn off the instrument before unplugging the power cord and or moving the instrument If the instrument is broken or dropped disconnect the power cord and contact a service person Do not disassemble the instrument Use only authorized accessories adaptor power cord and USB drive If the instrument emits smoke disconnect the power cord from the wall outlet and contact a service person A CAUTION Using cleaning or decontamination methods other than those recommended by the manufacturer may compromise the safety or quality of the instrument 1 CAUTION PHYSICAL INJURY HAZARD The instrument is to be serviced only by trained personnel or vendor specified in the user guide 40 Chemical safety Chemical hazard WARNING CHEMI
3. See page 7 for instructions on installing the instrument Visit www thermofisher com registercountess to register your instrument You will be asked to supply the serial number your name and your contact details Registering your instrument ensures that you will receive notifications of software upgrades and information on new assays for use with the Countess II Automated Cell Counter Instrument exterior components AAA H AHHH P Countess invi The trogen Pa O Touch screen display The 7 inch capacitive touch screen display is the main user interface of the Countess IT Automated Cell Counter It contains the buttons for all instrument functions and displays data from the cell count 2 5 USB ports The USB ports allow you to transfer and save the cell count data and image to an external computer for record keeping and printing purposes You can use the USB drive supplied with the instrument or any other standard FAT32 formatted USB drive for data transfer If desired you can plug in a USB mouse into the rear USB port for instrument control Note The USB ports located in the front and the back of the instrument function the same However the first USB drive connected will be the preferred saving location and both USB drives cannot be accessed at the same time 3 Slide port The slide port is used to insert the Countess Cell Counting Chamber Slide containing the sample into the instrument Power switch
4. level dry surface Remove the thin plastic protector film from the touch screen display Plug one end of the power cord appropriate for your region into the instrument Plug the power cord into the electrical outlet Be sure to use only the power cord supplied with your instrument Powering the instrument with an unapproved power cord may damage the instrument Turn ON the 1 Turn on the instrument by flipping the power switch on the back of the instrument instrument page 6 to the ON position The instrument initializes and displays the Home screen Countess Il Insert slide 2 From the Home screen you can proceed immediately to the assays by inserting a slide page 13 Alternatively you can change or add a profile Step 3 below or change instrument settings Step 4 below 3 To change the current profile or to add a new profile to the instrument gt press the Profiles button in the upper left corner Profiles allow you to create customized count preferences i e gate counts based on cell size brightness and or circularity page 9 4 To change instruments settings press the Instrument Settings button in the upper right corner Instrument settings allow you to update the Countess II software and change the date and time page 23 Load profile Profiles screen Profiles screen allows you to create and save up to 9 customized profiles Each custom profile defines the count parameters size bright
5. 2015 New user guide The guides listed below are available for the Countess II Automated Cell Counter Guide Pub no Countess II Automated Cell Counters User Guide MAN0014293 Countess II and Countess II FL Automated Cell MANO0010826 Counters Quick Reference Card QRC Additional resources are available on the Countess Technical Resources page Go to www thermofisher com countess to access protocols application notes and tutorials Text and keyboard conventions used in this user guide are listed below For safety alert words and symbols used in this document see page 4 Convention Use Bold Bold text indicates user action For example Press More gt Right arrow symbol gt indicates a menu choice and separates successive commands you execute or select from a drop down or shortcut menu For example Select More gt Adjust Two user attention words appear this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product T IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical Safety alert words Four safety alert words appear in this document at points where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particu
6. have a set point from which to focus on the cells in subsequent samples 1 Prepare the sample by adding 10 uL of cell suspension to 10 uL of 0 4 trypan blue stain Mix the sample mixture well by pipetting up and down a few times 2 Load 10 uL of the sample mixture into the Countess Cell Counting Chamber Slide page 13 Let the sample mixture settle for 30 seconds to ensure a uniform focal plane 3 Insert the sample slide into the slide port of the instrument making sure that the side containing the sample is inserted completely 4 When the slide is inserted the instrument automatically illuminates the sample sets the intensity of the brightfield light source and auto focuses on the cells Capture Mi SOU J Capture 29 9 To manually adjust the focus press the Focus button 6 Use the Focus slider or the plus and minus buttons to refine the brightfield focus Note If needed Zoom in on the image to adjust focus or lighting 7 After finding the optimal focus press Set to set the nominal focus Once the focus has been set the Set button on the focus slider becomes inactive confirming that the focus setting has been stored 30 E Sel Appendix A Troubleshooting Note The software for the Countess II is updated regularly If you are having any issues with your experiments first check the website to see if a new software version is available You can download the most recent version of t
7. shock always turn off the Countess II Automated Cell Counter and unplug the power cord before cleaning or decontaminating the instrument A N CAUTION All biological samples and materials that come into contact with them have the potential to transmit infectious diseases and are considered biohazardous Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Q IMPORTANT Using a cleaning or decontaminating method other than that s manufacturer may result in damage to the instrument e Wipe the touch screen of the Countess II Automated Cell Counter using a soft lint free cloth moistened with an LCD cleaning solution Do not apply excessive force during cleaning Wipe the touch screen dry immediately after cleaning e Ensure that the cleaning solution does not enter the power button the power inlet the slide port or the USB ports e Never pour or spray any liquids directly on the instrument to avoid electrical shock when the instrument is plugged in e Do not use abrasive cleaning solutions or material to prevent the touch screen from getting scratched e Wipe the instrument case of the Countess II Automated Cell Counter using a soft lint free cloth moistened with distilled water Wipe the instrument dry immediately after cleaning e Ensure that water or other cleaning solutions do not enter the power button the power inlet the slide port o
8. that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations gt Except for IMPORTANT safety alerts each safety alert word in the document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to the instrument see Safety symbols 36 Symbols on instruments Electrical symbols The following table describes the electrical symbols that may be displayed Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates the On Off position of a push push main power switch Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a terminal that can receive or supply alternating current or voltage Indicates a terminal that can receive or supply alternating or direct current or voltage 37 Safety symbols Environmental symbols The following table describes the safety symbols that may
9. this before proceeding If necessary reformat the USB drive to FAT32 following the recommended procedure for your operating system Note Reformatting the USB drive will result in the loss of all files Back up the files in the USB drive prior to reformatting e The software update file must be saved on the top level of the USB drive not within a folder or a subfolder e The software update file must be uncorrupted during transfer Do not rename zip or compress the software update file Update the 1 Go to www thermofisher com countessupdate and download the latest Countess II II FL Countess II II FL software version to your desktop software Note The software update file has a version specific name followed by the 2 Copy the software update file onto the USB drive making sure that it is saved on the top level and not hidden within a folder Insert the USB drive into one of the USB ports of the instrument see page 6 4 Press the Instrument Settings button on the Home page page 8 to open the Instrument Settings screen page 23 9 Select Software Update from the Instrument Settings menu The instrument scans the USB drive for the latest software version Software Update A software update Is available Would you like to update your software now Update Now 6 When prompted select Update Now 7 Once the update has completed restart the instrument 24 Date Time Set the date and 1 Press the I
10. with any third party software or spreadsheet program The table below describes the categories of the Countess II data saved as a CSV file and opened with a spreadsheet program ee e General Trypan Blue Brightfield General Details Trypan Blue Brightfield Count Parameters a fe fo fase arenes CF ra ur urbe f cel counednthasangla 8 icons Concentration af just th i partion ofthe ump 1 ead concentra Concentration o usa dent portion othe sargle ee roi pation numba Ty times rita grs ale from 100 o e Minium sic Ui canins ak ii max Mars ie of ells iran a migra in Live adjust sider velo for iia rasa 35 Appendix E Safety Safety conventions used in this document Safety alert words Four safety alert words appear in this document at points where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation
11. CAL HAZARD Before handling any chemicals refer warning to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions FIN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles General safety To minimize the hazards of chemicals guidelines e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See Documentation and support page 46 e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Comply with all local state provin
12. E TS SUITS us 20 Save cont LOS UU S a toa et taa 20 NS SELLING SSSR de sn nn a antenne tenue 23 ON a did asa eee eames 23 DOPE W ATU Pd AUC acess cabs ech ae ee OS 24 DA dm ete Reo teeta ne nt Pema nagar i noi li ates 25 Fs Mantencion 27 Instrument Careiro A 27 Clean the Countess II Automated Cell Counter ss 28 BSL MOM All OCU nsan a tele aaa a Or 29 Appendix A Troubleshooting cei ission nn ween ne encoun aaa a a 31 Appendix B Produet Speci lcations ec oi 33 Technical SOC CIEICAU ORNS aa N am teen on 33 Appendix C Ordering INTO Mato wasusia 34 Countess Il Aaitomated Cell Outer ere la aida 34 Accessory PE OCICS ta oss AAA NAN 34 Appendix DE COV TIS TO Malone 35 CSV LOT AE explained 35 APDPendI E aida 36 Safety conventions usediin this documenta 36 Symbol onin HUME S erene N 37 satety labels On A struments salidas 39 Generals ISIE SU NS 40 Ehemica Ste A eu ne 41 Ehemical waste satelites tii oi 42 A ns ctpsa ace hide do On Re Un 43 Biological hazard SOT Y en chy iant 44 Safety and electromagnetic compatibility EMC standards ss 45 WO CURIETILALIO I ANS CD DOCS O tenets eta a aeaa aaaea 46 OD rat SP PO den ira 46 Audience Revision history User documentation Text and keyboard conventions User attention words About this guide This user guide is for laboratory staff operating maintaining and analyzing data using the Countess IT Automated Cell Counter Revision Date Description A 0 August
13. The ON OFF rocker switch is the main power switch It is not necessary to use the power switch for day to day operation of the instrument 6 Power input jack The power input jack connects the instrument to an electrical outlet through the supplied power cord and the appropriate plug based on the electrical outlet configuration in your country dy Note The Countess II Automated Cell Counter Cat no AMQAX1000 uses brightfield illumination only and does not support the EVOS LED light cubes Installation Operating environment Install the instrument 2 Getting started Place the instrument on a level surface away from vibrations emanating from other pieces of equipment Allow at least 5 cm 2 in free space at the back of the instrument to allow for proper ventilation and prevent overheating of electronic components Set up the instrument away from direct light sources such as windows Ambient room lighting can enter the imaging path and affect the image quality Operating temperature range 4 32 C 40 90 F Relative humidity range 30 90 Q IMPORTANT Do not position the instrument so that it is difficult to turn off the main power switch located on the back of the instrument see page 6 In case of an instrument malfunction turn the main power switch to the OFF position and disconnect the instrument from the wall outlet NE Unpack the instrument and place the instrument on a flat
14. USER GUIDE invitrogen Countess Il Automated Cell Counter For brightfield applications Catalog Number AMQAX1000 Publication Number MANO014293 Revision A 0 ThermoFisher For Research Use Only Not for use in diagnostic procedures SCIENTIFIC Information in this document Is subject to change without notice DISCLAIMER TO THE EXTENT ALLOWED BY LAW LIFE TECHNOLOGIES AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified 11 Contents ABOUTIT GUNG iii 3 1 Product INTOFMALION ee detailed nent 5 Product aese py HOME Re AR ol 5 Instrument exterior components eaa R A 6 Z GOING Sa Ed anana aaa 7 STATION A RNA E AN 7 Load Droles amendement O 9 3 Cell COUNE and cellviability ASS Sinai 13 PEED al SSATP Coss ee E ON de ot en ten 13 COUR COS ee eer ee a ee ne ae 14 i RESUS il 16 NS ii 16 dentitythe cl comedo 17 Crap COUNT ES Still 18 Gale COUN Fe SUES OREIRO PAE Po e en ne a te ee shone dc aes T 19 9s SAV
15. be displayed Each symbol may appear by itself or in combination with text that explains the relevant hazard see Safety labels on instruments These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol PEPEE LS gt Description Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution Indicates the presence of a laser inside the instrument and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of an ultraviolet light and to proceed with appropriate caution The following symbol applies to all Thermo Fisher Scientific electrical and electronic products placed on the European market after August 13 2005 Symbol i l Description Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of waste electrical and electronic equipment WEEE European Union customers Call you
16. bility Make sure that you do not shake or agitate the slide between counts Abnormally high percentage of dead cells or live cells counted as dead Ensure that the cells are focused correctly so that live cells have bright centers and dead cells are dark throughout see page 14 If the cells are not well focused and look dark on the screen the Countess II will count them as dead cells If cells are well focused have bright centers and are being counted as dead confirm that they are within the appropriate cell size range and try adjusting the settings If cells are exposed to trypan blue for a long period of time viability could be affected so slide should be prepared and counted fresh each time for best results Gate out the debris using the size brightness and circularity sliders USB drive not recognized by the instrument The USB drive must be FAT32 formatted to be recognized by the instrument If it is not reformat the USB drive to FAT32 page 24 Try another correctly formatted USB drive Unable to update the Countess software Make sure the USB drive is formatted to FAT32 If it is not reformat the USB drive to FAT32 before transferring the files onto the USB drive for software update The update file must sit on the top level of the USB drive not within a folder or a subfolder File cannot be renamed in any way File cannot be zipped or compressed during distribution It must be uncorru
17. cial or national laws and regulations related to chemical storage handling and disposal 41 Chemical waste safety Chemical waste hazard CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets SDSs and local regulations for handling and disposal Chemical waste To minimize the hazards of chemical waste safety guidelines Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local sta
18. d on cell size or fluorescence intensity make sure that the desired graph is displayed on the Results screen Save procedure 1 To save your data insert the Countess II USB drive or equivalent into an available USB port on the instrument see page 6 Note The USB ports located in the front and the back of the instrument function the same However the first USB drive connected will be the preferred saving location 2 On the Results screen press Save to navigate to the Save screen File name DDBA Study45 lu Py Result Images JPG JPG DDBA_Study45 jpg DDBA_Study45_BRT jpg DDBA_Study45 csv 20 3 To assign a name to your experiment press the File name File name text field The alpha numeric keypad opens 4 Enter the file name using the alpha numeric keypad To enter symbols press the symbol amp key To return to the alpha numeric keypad press ABC Insert name x DDBA Study45 5 Press Enter to save the name and return to the Save screen x To return to the Save screen without saving the name press the close button 6 Select the desired mode s to save your experiment Result Images Data You can select an individual mode e g Result only or any combination of modes e g Result Images and Data In the example below Images and Data are selected File name DDBA Study45 Result Images DDBA_Study45 jpg DDBA_Study45_BRT jpg DDBA_Study45 csv Cancel Perr
19. er com or contact Technical Support page 46 Countess II Automated Cell Counter AMQAX1000 Countess II power cord with four adapter AMEP4716 cords Countess II USB drive A25751 Countess II Disposable Slide Holder AMEP4745 Accessory products The following products can be used with the Countess II Automated Cell Counter and are available separately from Thermo Fisher Scientific For more information visit www thermofisher com or contact Technical Support page 46 Countess Cell Counting Chamber Slides 1 box C10228 50 Slides 100 counts Countess Cell Counting Chamber Slides 10 boxes C10312 500 Slides 1000 Counts Countess Cell Counting Chamber Slides 25 boxes C10313 1250 Slides 2500 Counts Countess Cell Counting Chamber Slides 50 boxes C10314 2500 Slides 5000 Counts Countess Cell Counting Chamber Slides 100 boxes C10315 5000 Slides 10 000 Counts Countess Test Beads 1 x 10 beads mL C10284 Trypan blue stain 0 4 T10282 Each box of Countess Cell Counting Chamber Slides contains 50 slides and 2 x 1 mL vials of trypan blue 0 4 sufficient for 100 counts 34 Appendix D CSV file format CSV file format explained Overview A comma separated values CSV file stores tabular data numbers and text in plain text form Plain text means that the file is a sequence of characters with no data that has to be interpreted as binary numbers A CSV file can be opened
20. ffect the concentration reading e Vortex the bead stock on high for a full 30 seconds to resuspend and add 10 uL of the bead suspension to 10 uL of trypan blue without delay e Pipet the bead and trypan blue mixture up and down several times to make sure it is well mixed and immediately load 10 uE into the slide Variable counts for the same sample of cells e If you are pipetting different samples from the same cell sample the variability could be due to pipetting or mixing e Use recently calibrated pipettors and make sure that the cells are well suspended by pipetting up and down several times before adding trypan blue e Pipet the bead and trypan blue mixture up and down several times to make sure it is well mixed and load 10 uL into the slide without delay 31 Problem Possible solutions Variable counts when performing replicate counts of the same slide If you are counting replicates of the exact same slide visually inspect that all cells are counted correctly in the image There may be a slightly different field of view each time a slide is inserted Depending on the concentration and uniformity of the cells this will cause some variability when performing replicate counts of the same slide although it should be less than 10 When counting fewer cells a small field of view change for only a small number of cells can have a larger affect Count cells at a higher concentration to reduce varia
21. ghtness and circularity sliders adjust the count parameters Note For a description of the count parameters and count parameter controls i e parameter sliders see page 10 7 When finished press Done to save the changes to count parameters and return to the Advanced screen Press Cancel to return to the Results screen without saving the changes 8 On the Advanced screen press Count to recalculate your results with the new count parameters 9 To permanently save your results to an external computer using a USB flash drive see page 20 19 5 Save results Save count results Save screen The Countess II FL Automated Cell Counter allows you to save your data and images using a USB flash drive To save your experiment you can choose from the following options in any combination e Result Saves the Results screen as it is displayed on the instrument with or without the Graph in the selected image format JPEG BMP PNG or TIFF e Images Saves only the raw captured image in the selected image format JPEG BMP PNG or TIFF e Data Saves the data from the experiment as a CSV file comma separated values The CSV format allows for processing or re displaying results with any third party software or spreadsheet program For more information on the CSV file format see Appendix D CSV file format page 35 dy Note If you wish to save your results with the Graph showing the distribution of cells base
22. he software from www thermofisher com countessupdate You can also register your Countess II instrument at www thermofisher com registercountess to be informed of any future software updates Problem Possible solutions Autofocus does not seem to focus on the cells very well e Make sure there are no bubbles or debris visible on the screen that could interfere with the autofocus and make it more difficult to get the sample in the correct focal plane e Ideally the live cells should have bright centers compared to the dead cells which are dark throughout page 14 e Setting the nominal focus will improve autofocus consistency with future slides To set the nominal focus see page 29 Some cells appear on the image but are not included in the count e Adjust the size brightness and circularity gates for both live and dead cells to include all of the cells in the count page 18 e After including all of the cells in the count you can narrow the count criteria if you wish to exclude cells of a certain size or certain brightness e When the gates are fully maximized the CSV should indicate 0 60 for cell size and 0 255 for brightness Images are very bright and washed out Enable Auto Lighting from the Profiles menu or decrease the bright field light intensity before counting the cells Getting incorrect concentration for the Countess test beads e The beads can settle quickly in solution which will a
23. hen calculating the total cell concentration The cell concentration displayed in the Results screen is the original cell concentration before dilution into trypan blue 16 Identify the cells counted Advanced screen Identify Live and Dead cells The Advanced screen allows you to identify the objects i e cells counted in each channel and included in the count results for further review After reviewing the marked objects you can adjust the threshold for size brightness and or circularity as desired for your application 1 On the Results screen click More The Advanced screen opens Advanced Pp Default Adjust Zoom Graph 2 To identify the cells that are included in the count as live press the Live button Live cells will be circled in green on the screen To identify the cells that are included in the count as dead press the Dead button Dead cells will be circled in red on the screen Note You may select either or both options In the example below both Live and Dead buttons are pressed and live and dead cells are marked with green and red circles around them respectively 3 To unmark the cells identified as live green or dead red on the screen press the Live or the Dead button again respectively 17 Graph count results View graph For cell count and cell viability assays you can view the distribution of cells live and or dead based on s
24. ingle channel that is selected via the channel selection radio buttons e Size As you move the slider up the algorithm includes larger objects in the count As you move the slider down only the smaller objects are counted fe larger objects iF smaller objects e Brightness As you move the slider up the algorithm includes the brighter objects in the count As you move the slider down only the dimmest of objects are counted E3 brighter objects Es dimmer objects e Circularity As you move the slider up the algorithm includes more objects with shapes other than circular in the count As you move the slider down only the objects that are perfect circles are counted ia less circular B more circular e Size and brightness sliders are range sliders To adjust the upper and lower boundaries without changing the data range drag the slider by its middle section i e the slider bar To adjust only the upper or the lower boundary move the upper or the lower handle in the desired direction This will also change the range of values within which the cells are counted e The circularity slider only sets a single threshold value cells that fall below the set value are counted and cells that are beyond this range are excluded To adjust the threshold for circularity drag the slider in the desired direction 10 Load a profile Add edit a profile Press the Profiles button located on the upper left corner of the screen t
25. ining support Technical support Limited product warranty Visit www thermofisher com support for the latest in services and support including e Worldwide contact telephone numbers e Product support including Product FAQs Software patches and updates e Order and web support e Product documentation including User guides manuals and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 46 For support visit thermofisher com support Thermo Fisher thermofisher com SCIENTIFIC 1 September 2015
26. ize in a graphical format Note You can view the Graph on Results Advanced and Adjust screens 1 To view the graph showing the distribution live and or dead cells based FN on cell size press the Graph button Q M Zoom Graph 2 To view the distribution of only the live or dead cells check the corresponding Live or Dead check box on the graph The graph will automatically update and display the distribution of cells based on size only in the selected population 3 Optional Using the size brightness and circularity sliders adjust the count parameters As you adjust the count parameters the count results and the graph will be automatically updated 4 To close the graph press the Graph button again 18 Gate count results Adjust screen Gate count results The Adjust screen contains the controls for gating count results based on size brightness and circularity You can adjust the count parameters before or after performing a count 1 On the Results screen press More to open the Advanced screen 2 Optional Press the Live and or the Dead button to identify the cells in the selected population see page 17 3 Onthe Advanced screen press Adjust to open the Adjust screen 4 Optional Press the Graph button to view the distribution of cells live and or dead based on size as you gate the count results see page 18 Select the channel Live or Dead you wish to gate 6 Using the size bri
27. lar level of observation or action as defined below IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations gt Except for IMPORTANT safety alerts each safety alert word in this document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to the instruments see Safety symbols in Appendix E 1 Product information Product description Product contents Countess Il Automated Cell Counter Upon receiving the instrument Register your instrument The Countess II Automated Cell Counter is shipped with the components listed below Component Quantity Countess Automated Cell Counter Cat no AMQAX1000 1 each Power Cord with 4 adaptor cords 1 each for U S Canada Taiwan Japan Europe or UK Countess Cell C
28. lide is pushed in correctly 2 When the slide is inserted the instrument automatically illuminates the sample sets the intensity of brightfield illumination and auto focuses on the cells Note To turn off the Auto Lighting function see page 10 Capture Ol gt Capture Zoom Focus 3 Optional Press the Profiles button and load the desired profile amp Default as described on page 11 4 Optional If desired press the Focus button and then use the Focus slider to manually bring your sample into focus see Note below If needed Zoom in on the image while adjusting the focus hy Note The auto focus algorithm is designed to highlight the differences between live and dead cells Therefore the optimal focus level is where the live cells have a light colored center and the dead cells are dark throughout To enable optimal auto focus functionality you may need to initially refine the focus by adjusting it manually and then setting it as the nominal focus For more information see page 29 9 Optional Manually adjust exposure using the light source slider The light source slider controls the LED intensity camera gain and exposure time and it is used for adjusting the image brightness 14 6 Press Capture The instrument temporarily captures the image and displays the results total concentration percentage and concentration of live and dead cells For more information see Results
29. ness and circularity and the Auto Lighting function for a consistent and streamlined workflow Default Small Cell Empty Empty Empty e You can access the Profiles screen from the Home Capture Results Advanced or Adjust screens e The current profile is displayed on the upper left corner of the Home Capture Results Advanced or Adjust screens e The Auto Lighting function and count parameters are set in the Edit profile screen page 10 e The Default profile contains default count settings and cannot be edited e The count parameters specified in the selected profile are applied to all new cell counts e If you have already performed a count loading a new profile from the Results screen applies the count preferences to the current counts results total cells viability etc and to all new counts Edit profile screen The Auto Lighting function and count parameters are set in the Edit profile screen Edit profile Profile name Trypan C Count settings E Auto FL Threshold E Auto Lighting Auto Lighting Auto Lighting function automatically illuminates the sample in the brightfield channel for increased sample to sample consistency and decreased user to user variability By default the Auto Lighting function is turned on to toggle it on and off use the Auto Lighting checkbox see page 11 Count parameters Count parameters are set using the parameter sliders which correspond to a s
30. nstrument Settings button on the Home page page 8 to open time the Instrument Settings screen 2 Press Date Time on the Instrument Settings menu to open the Date Time screen Date Time Date O MM DD YYYY o DD MM YYYY 12Hour ORGA 0 3 Select the Date format you wish to use 4 Press any Date text box MM DD or YYYY to open the Edit Date keypad Edit Date Month 9 Using the keypad enter the date into Month Day and Year text boxes pressing Enter after each entry 6 After you are finished entering the date press the close button to return to the Date Time screen 25 7 Select the Time format you wish to use Available options are 12 Hour and 24 Hour formats Date Time Time 12Hour KN EH 0 o Cancel Se o 8 Press any Time text box Hours or Minutes to open the Edit Time keypad Edit Time Eu Minutes 9 Using the keypad enter the time into Hours and Minutes text boxes pressing Enter after each entry 10 After you are finished entering the time press the close button to return to the Date Time screen 11 If you have selected the 12 Hour format select AM or PM 12 Press Save to set the Time and Date and return to the Instrument Settings screen Press Cancel to return to the Instrument Settings screen without saving your changes 26 7 Maintenance Instrument care General guidelines Use the appropriate cleaning solutions for each componen
31. o gt open the Profiles screen Press the desired profile to select and then press Load The instrument will load the count parameters specified in the selected profile and return to the previous screen To return to the previous screen without loading the new profile press the previous button The instrument will keep the saved profile but return to the previous screen without loading it Press the Profiles button located on the upper left corner of the screen to gt navigate to the Profiles screen To add or edit a new profile select an empty or an existing profile and then press Edit The Edit screen for the selected profile opens Note The Default profile contains default count settings and cannot be edited Edit profile Trypan whe pefautt eee Count settings Auto FL Threshold Auto Lighting Select or deselect the Auto Lighting checkbox to turn the Auto Lighting function ON or OFF To define the new count parameters select Live or Dead from the Trypan Blue radio buttons Adjust the size brightness and circularity thresholds using the appropriate parameter slider page 10 dy Note Although the Auto FL Threshold checkbox and the FL channel radio buttons are visible on the Edit profile screen they are not functional on Countess II Automated Cell Counters which only feature a single brightfield channel 11 6 To assign a name to the new profile or to change the name of the e
32. ounting Chamber Slides 50 slides box 1 box Countess ll Disposable Slide Holder 1 each Countess Il USB drive 1 each Countess Il Automated Cell Counter Quick Reference Card 1 each The Countess II Automated Cell Counter is a fully automated cell counter and assay platform that uses state of the art optics and image analysis algorithms to analyze trypan blue stained cells in suspension e The Countess II Automated Cell Counter offers an intuitive user interface and provides the option to save data and generate a report which can then be transferred to a PC using the USB drive supplied with the instrument or available separately e The cells to be counted are loaded into the instrument in disposable Countess Cell Counting Chamber Slides page 13 Each chamber slide contains two enclosed chambers to hold the sample to allow you to measure two different samples or perform replicates of the same sample e The Countess II Automated Cell Counter takes 10 seconds per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells In addition to cell count and viability the instrument also provides information on cell size Examine the instrument carefully for damage incurred during transit Ensure that all parts of the instrument including accessories listed above are included with the product Damage claims must be filed with the carrier the warranty does not cover in transit damage
33. page 16 Results Total concentration 1 50 x 10 mL Trypan Blue dilution corrected LIVE 70 1 04 x 10 mL DEAD 30 4 52 x 10 mL Q M Zoom Graph Next steps e To identify the objects i e cells counted as live or dead press More to navigate to the Advanced screen page 17 e To see the distribution of live and dead cells in a graphical format press the Graph button see page 18 e To gate the results by object size brightness or circularity first press More to open the Advanced screen and then press Adjust to navigate to the Adjust screen page 19 e To permanently save the results press Save see page 20 e To perform anew count remove the slide and reinsert it into the instrument to count the sample in the second chamber 15 4 Results View results The Results screen for cell count and cell viability assays displays a composite image of the objects counted and the results of the cell count and cell viability calculations total concentration percentage and concentration of live and dead cells Results screen Results Total concentration 1 50 x 10 mL Irypan Blue lilution corrected LIVE 10 1 04 x 10 mL DEAD 30 4 52 x 10 mL Q M Zoom Graph dy Note When performing brightfield BF cell counts and cell viability assays the counting algorithm assumes that you have diluted your cells 1 1 in trypan blue and takes this dilution into account w
34. ply in your facility gt gt gt gt DANGER ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity The Countess II Automated Cell Counter has an installation overvoltage category of Il and is classified as portable equipment 43 Biological hazard safety a WARNING BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications ATTENTION BIOHAZARD Les chantillons biologiques tels que les tissus les fluides corporels et le sang des humains et d autres animaux ont la possibilit de transmettre des maladies infectieuses Suivre tous les r glements municipaux provinciaux provincial et ou nationales en vigueur Porter des lunettes de protection appropri des v tements et des gants In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 www cdc gov OD ohs biosfty bmb14 bmbl4toc htm Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution
35. pted during transfer and have a lft suffix If needed check that the USB port is functional by testing a USB mouse 32 Technical specifications Physical characteristics Technical specifications Optics Analysis slide Instrument type Instrument dimensions Weight Operating power Frequency Electrical input Installation site Operating temperature Operating humidity Processing time Sample concentration range Particle cell diameter range Required sample volume Firmware USB Drive Optics Camera Material Dimensions Chamber volume 33 Appendix B Product specifications Benchtop cell counter and suspension cell based assay platform 9 W x 5 D x 9 inches H 8 lbs 100 240 VAC 0 58 A MAX 50 60 Hz 12 VDC 2 A Indoor use only Class A Environments i e non residential or light industrial Pollution degree 2 10 40 C lt 80 non condensing 15 seconds 1 x 1041 x 107 cells mL 4 60 um particles 7 60 um cells 10 uL Countess Automated Cell Counting Platform Software 4 Gigabyte 1 channel brightfield 5 Mega pixels 2 5x Optical Magnification Poly methyl methacrylate PMMA 25 mm W x 75 mm D x 1 7 mm H 10 uL Appendix C Ordering information Countess I Automated Cell Counter The Countess II instrument and instrument accessories are available from Thermo Fisher Scientific For more information visit www thermofish
36. r Customer Service representative for equipment pick up and recycling See www thermofisher com for a list of customer service offices in the European Union 38 Safety labels on instruments The following CAUTION WARNING and DANGER statements may be displayed Thermo Fisher Scientific instruments in combination with the safety symbols described in the preceding section Hazard symbol English Francais ZN CAUTION Hazardous chemicals Read the Safety Data Sheets SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es a la manipulation et l limination des d chets N DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Thermo Fisher Scientific qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune piece r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de ser
37. r the USB ports e Never pour or spray any liquids directly on the instrument to avoid electrical shock when the instrument is plugged in e Wipe the instrument case of the Countess II Automated Cell Counter using a soft lint free cloth moistened with 70 alcohol Wipe the instrument dry immediately after cleaning e Avoid using a bleach solution because it may leave a residue of bleach crystals on the instrument e Ensure that water or other cleaning solutions do not enter the power button the power inlet the slide port or the USB ports e Never pour or spray any liquids directly on the instrument to avoid electrical shock when the instrument is plugged in 28 Set nominal focus Overview Set nominal focus Nominal focus is the Z point i e depth around which the auto focus function searches to provide fine focus to the sample The auto focus algorithm of the Countess II FL Automated Cell Counter is designed to highlight the differences between live and dead cells in the brightfield channel The optimal focus level is where the live cells have a light colored center and the dead cells are dark throughout see examples below Erap o 0 MENT 2 o Focus is not optimal Focus is optimal To enable optimal auto focus functionality you may need to initially refine the brightfield focus by adjusting it manually and then setting the nominal focus This allows the auto focus function to
38. s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check your local guidelines and legislation on biohazard and biosafety precaution and the best practices published in the World Health Organisation WHO Laboratory Biosafety Manual third edition www who int csr resources publications biosafety WHO_CDS_CSR_LYO_ 2004_11 en 44 Safety and electromagnetic compatibility EMC standards U S and Canadian safety standards European safety and EMC standards CE Australian EMC standards This section provides information on e U S and Canadian safety standards e European safety and EMC standards e Australian EMC standards The CSA C US Mark signifies that the product meets applicable U S and Canadian standards including those from CSA CSA America ANSI ASME ASSE ASTM NSF and UL The CE Mark symbolizes that the product conforms to all applicable European Community provisions for which this marking is required Operation of the instrument is subject to the conditions described in this manual The protection provided by the instrument may be impaired if the instrument is used in a manner not specified by Thermo Fisher Scientific The C Tick Mark indicates conformity with Australian and New Zealand standards for electromagnetic compatibility 45 Documentation and support Obta
39. t as indicated in the for care cleaning procedures on page 28 e If liquid spills on the instrument turn off the power immediately and wipe dry Power supply Always use the correct power supply The power adaptor specifications appear on the serial number label bottom of the instrument and in the Technical specifications section of this user guide page 33 Damage due to an incompatible power adaptor is not covered by warranty CAUTION Never disassemble or service the instrument yourself Do not remove any covers or parts that require the use of a tool to obtain access to moving parts Operators must be trained before being allowed to perform the hazardous operation Unauthorized repairs may damage the instrument or alter its functionality which may void your warranty Contact your local distributor to arrange for service 1 IMPORTANT If you have any doubt about the compatibility of decontamination or cleaning agents with parts of the equipment or with material contained in it contact Technical Support page 46 or your local distributor for information 27 Clean the Countess Il Automated Cell Counter Introduction Clean the touch screen Clean the instrument case Decontaminate the instrument We recommend cleaning the Countess IT Automated Cell Counter periodically to prevent the buildup of dust and dirt that might reduce its performance and cause contamination PAS CAUTION To avoid electrical
40. t the cells immediately after staining per the assay protocol Do not press the optical surfaces of the chamber slides Hold the slides by the edges Take care to avoid forming bubbles in the sample Prepare the sample by adding 10 uL of your cell suspension to 10 uL of 0 4 trypan blue stain Mix the sample mixture well by pipetting up and down a few times Gently pipet 10 uL of the trypan blue stained sample into the half moon shaped sample loading area The sample is loaded into the chamber through capillary action mm CO m aa E Note Each chamber in the Countess Cell Counting Chamber Slide has a 10 uL sample capacity Do not overfill the slide chambers Let the sample mixture settle in the chamber for 30 seconds and then insert the slide into the slide port see page 6 You will hear a soft click if the slide is pushed in correctly To remove the slide push the slide gently into the instrument until it clicks and a spring pushes the slide out Grasp the slide and pull it out the rest of the way amp Note After using the Countess Cell Counting Chamber Slides appropriately dispose of them as biohazardous waste Do not reuse the disposable chamber slides 13 Count cells Count procedure 1 Load the Countess Cell Counting Chamber Slide with your trypan blue stained sample and insert it into the slide port of the instrument as described on page 13 You will hear a soft click if the s
41. te provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply 42 Electrical safety Fuses Power Overvoltage rating gt DANGER ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the Countess II Automated Cell Counter without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument DANGER ELECTRICAL HAZARD Grounding circuit continuity is vital for the safe operation of equipment Never operate equipment with the grounding conductor disconnected DANGER ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage sup
42. vice qualifi venant de Thermo Fisher Scientific DANGER Class 3B visible and or invisible laser radiation present when open Avoid exposure to beam DANGER Rayonnement visible ou invisible d un faisceau laser de Classe 3B en cas d ouverture Evitez toute exposition au faisceau gt gt CAUTION Moving parts Crush pinch hazard ATTENTION Pieces en mouvement risque de pincement et ou d crasement 39 General instrument safety Operating the instrument Safety precautions Cleaning or decontaminating the instrument Removing covers or parts of the instrument WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Thermo Fisher Scientific may result in personal injury or damage to the instrument Ensure that anyone who operates the instrument has Received instructions in both general safety practices for laboratories and specific safety practices for the instrument Read and understood all applicable Safety Data Sheets SDSs See Documentation and support page 46 Do not install the instrument in heavy humidity such as a greenhouse or an incubator to avoid a danger of electric shock If water or other material enters the instrument the adaptor or power inlet disconnect the power cord and contact a service person For operating environment refer to Operating environment page 7
43. xisting profile press the Profile name text box The alpha numeric keypad opens Insert name x DDBA Study45 7 Type in the desired profile name using the alpha numeric keypad To enter symbols press the symbol amp key To return to the alpha numeric keypad press ABC 8 Press Enter to save the name and return to the Edit profile screen To return to the Edit profile screen without saving the name press the x close button 9 Press Save to save the new profile and then press Close in the confirmation screen to return to the Profiles screen To return to the Profiles screen without saving press Cancel 10 On the Profiles screen press Load The instrument will load the count parameters specified in the selected profile and return to the previous screen 11 To return to the previous screen without loading the new profile press the previous button The instrument will keep the saved profile but return to the previous screen without loading it 12 Prepare sample Recommendations Load Countess Chamber Slide 3 Cell count and cell viability assays To obtain the best results follow these recommendations Ensure that the cell sample is homogeneously mixed The measurement range extends from 1 x 10 1 x 10 cells mL but the optimal range is 1 x 10 4 x 10 cells mL For accurate results in cell viability assays ensure that the counting area is covered with the cell suspension and coun
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